The reactions were run through the following programme: 95°C for 10 min, followed by 40 cycles at 95°C for 15 sec and 60°C for 1 min. Further, specificity was assessed by the melting curves (95°C for 15 sec, 60°C for 1 min, and 97°C continuous).
Chromo4 real-time PCR machine (Biorad) was used with the following programme: 95 °C for 10 minutes, 95 °C for 10 seconds, 60 °C for 15 seconds, 72 °C for 15 seconds, 75 °C for 1 second, plate read, repeat from the second step 39 more times, 72 °C for 5 minutes, melting curve from 55 °C to 95 °C, read every 0.2 °C, hold for 1 second and finally 75 °C for 5 minutes.
Amplification was performed using a Dice PCR Thermal Cycler (TaKaRa Bio) with the following programme: initial denaturation for 2 min at 94°C, followed by 35 cycles of denaturation for 10 s at 98°C and annealing and extension for 2 min at 68°C.
The magnesium chloride concentrations for each primer pair were 2.5 mM (GAPDH) and 1.25 mM (aggrecan), and the following programme was used for aggrecan reactions: 94°C for 3 minutes, 24 cycles of 94°C for 1 minute, 60°C for 1 minute, and 72°C for 1 minute 30 seconds.
Real-time quantitative polymerase chain reaction (PCR) was carried out using a StepOnePlus Real-Time System (Applied Biosystems, Mannheim, Germany) with the following programme: activation at 95 °C for 20 s followed by 50 cycles of 95 °C for 1 s and 60 °C for 20 s.
In short, a single colony was added to 20 μL InstaGene Matrix (Bio-Rad) and in a thermal cycler the following programme was run: 56°C for 10 min, 99°C for 5 min, and 4°C forever.
The following programme was used for all reactions: 95°C 15 min → 45 × (95°C 15 s → 60°C 1 min).
A PCR amplification system (PTC-100: MJ, USA) was used with the following programme: initial denaturation for 4 min at 94°C, 30 s at 94°C, 30 s at 56°C, 30 s at 72°C, 20 cycles; 7 min at 72°C.
The PCR amplification was performed using the Roche Lightcycler 480 Real-time PCR instrument and the SYBR Green Realtime PCR Master Mix (Toyobo, Japan) with the following programme: 95°C denaturation for 30 s, and then 45 cycles of denaturation at 95°C for 5 s, annealing at 54°C for 10 s, elongation at 72°C for 15 s.
The amplification was performed with the following programme: 40 cycles of 95°C for 15 s, and 60°C for 60 s.
